CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel (CDC Flu rrt-pcr Dx Panel)

CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel (CDC Flu rrt-pcr Dx Panel) Influenza A/B Typing Kit Instructions for Use Package Insert Catalog # FluIVD03-1 1000 reactions For In-vitro Diagnostic (IVD) Use Rx only Centers for Disease Control and Prevention Influenza Division 1600 Clifton Rd NE Atlanta GA 30333

Table of Contents Intended Use... 2 Summary and Explanation... 2 Principles of the Procedure... 5 Materials Required (Provided)... 7 Materials Required (But Not Provided)... 8 Warnings and Precautions... 10 Reagent Storage, Handling, and Stability... 11 Specimen Collection, Handling, and Storage... 12 Specimen Referral to CDC... 12 Reagent and Controls Preparation... 13 General Preparation... 15 Assay Setup... 16 Interpretation of Results and Reporting... 35 Influenza A/B Typing Assay Results Interpretation Guide... 37 Standards-Based Electronic Laboratory Reporting for Influenza... 38 Quality Control... 39 Limitations... 39 Performance Characteristics... 41 Disposal... 53 References... 53 Contact Information, Ordering, and Product Support... 55

Intended Use The Influenza A/B Typing Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rrt-pcr) assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR instrument in conjunction with clinical and epidemiological information: For qualitative detection of influenza virus type A or B viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NPS], nasal swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/throat swabs [NPS/TS]) and lower respiratory tract specimens (including bronchoalveolar lavage [BAL], bronchial wash [BW], tracheal aspirate [TA], sputum, and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture. To provide epidemiologic information for surveillance of circulating influenza viruses. Performance characteristics for influenza were established during a season when seasonal influenza viruses A/H1 and A/H3 were the predominant influenza A viruses in circulation and during a season when the A/H1pdm09 influenza virus was the predominant influenza A virus in circulation. Performance characteristics may vary with other emerging influenza A viruses. Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. Conversely, positive results do not rule out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens. All users, analysts, and any person reporting results from use of this device should be trained to perform and interpret the results from this procedure by a competent instructor prior to use. CDC Influenza Division will limit the distribution of this device to only those users who have successfully completed a training course provided by CDC instructors or designees. Summary and Explanation Influenza viruses are the causative agents of influenza infection, an acute respiratory illness that is highly contagious and results in significant morbidity each year with flu-related deaths ranging from a low of about 3,000 to a high of about 49,000 people (http://www.cdc.gov/flu/about/disease/symptoms.htm). Influenza virus types A and B primarily infect 2

the nasopharyngeal and oropharyngeal cavities. Influenza A viruses are further categorized into subtypes based on two major surface proteins, hemagglutinin (HA) and neuraminidase (NA). Similarly, Influenza B viruses can also be separated into two major lineages. The infective potential of influenza is frequently underestimated and can result in high morbidity and mortality rates, especially in elderly persons and in high risk patients, such as the very young and immunocompromised. Typical influenza viral infections in humans have a relatively short incubation period of 1 to 2 days, with symptoms that last about a week including an abrupt onset of fever, sore throat, cough, headache, myalgia, and malaise. When a subject is infected with a highly virulent strain of influenza, these symptoms can progress rapidly to pneumonia and in some circumstances death. Pandemic outbreaks of highly virulent influenza present a serious risk to human and animal health worldwide. Influenza viruses may be detected and characterized from clinical specimens by several methods that vary in sensitivity, speed, and capability with regard to distinguishing influenza types and subtypes. The CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel consists of nucleic acid amplification assays that detect influenza A and B viruses and further characterizes influenza A subtypes A/H1, A/H1pdm09, A/H3, and A/H5 (Asian lineage) and influenza B lineages B/Victoria and B/Yamagata. Components of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel can be obtained in several kit configurations. CDC Human Influenza Real Time RT PCR Diagnostic Panel FluIVD03 Kit Catalog # Contents Application FluIVD03 1 A/B Typing Kit Primer and Probe Sets: InfA, InfB, RP Controls: Pooled Influenza Positive Control (PIPC), Human Specimen Control (HSC) Detects all influenza A and B viruses FluIVD03 2 A Subtyping Kit FluIVD03 3 A/H5 Subtyping Kit FluIVD03 4 B Lineage Genotyping Kit Primer and Probe Sets: InfA, H1, H3, pdminfa, pdmh1, RP Controls: PIPC Primer and Probe Sets: InfA, H5a, H5b, RP Controls: H5VC, HSC Primer and Probe Sets: InfB, VIC, YAM, RP Controls: Influenza B Positive Control (IBPC) Specifically detects and differentiates seasonal influenza subtypes; H1, H3, H1pdm09 Detects classical swine influenza viruses and swine triple reassortant viruses 1 Specifically detects influenza A/H5 Asian lineage viruses Specifically detects and differentiates influenza B lineage genotypes; B/Victoria, B/Yamagata 1 Classical and triple reassortant swine viruses are not seasonal viruses and must be referred to CDC for further confirmation 3

Laboratories may use the influenza A, B, A/H1, A/H1pdm09, and A/H3 and A/H5 (Asian Lineage) primer and probe sets simultaneously to type and/or subtype suspected influenza positive clinical specimens. Additionally, influenza B viruses may be further characterized as B/Victoria or B/Yamagata using the VIC and YAM primer and probe sets. The CDC Human Influenza Virus Real- Time RT-PCR Diagnostic Panel assists in routine surveillance of seasonal influenza following an algorithm as follows: 4

Principles of the Procedure The Influenza A/B Typing Kit contains components of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel that is used in rrt-pcr assays on the ABI 7500 Fast Dx Real-Time PCR Instrument. The primer and probe sets contained in the Influenza A/B Typing Kit are designed for the detection of influenza type A and B viruses that infect humans. The Influenza A/B Typing Kit consists of oligonucleotide primers and dual-labeled hydrolysis (TaqMan ) probes and controls which may be used in rrt-pcr assays for the in vitro qualitative detection and characterization of the human influenza virus RNA in respiratory specimens from patients presenting with influenza-like illness (ILI). The oligonucleotide primers and probes for detection of Influenza A and B viruses were selected from highly conserved regions of the matrix (M) and non-structural (NS) genes, respectively. Detection of viral RNA not only aids in the diagnosis of illness caused by seasonal, newly emerging, and novel influenza viruses in patients with ILI, but also provides epidemiological and surveillance information on influenza and aids in the presumptive laboratory identification of specific novel influenza A viruses. 5

Summary of Influenza A/B Typing Kit Process Extract PIPC RNA Upon receipt of rrt-pcr Flu Panel reagents Resuspend primers and probes; aliquot and store Dilute PIPC, RNA 1:10, aliquot and store Upon obtaining sample Extract sample RNA and HSC RNA Prepare master mix (20 L) Prepare rrt-pcr plate (5 L RNA) Run assay on ABI 7500Fast Dx Analyze data Report results 6

Materials Required (Provided) Influenza A/B Typing Kit Contents: Catalog # FluIVD03-1 Box #1: Primers and Probes Reagent Label Part # Description Quantity / Tube Reactions / Tube InfA-F SO3304 Influenza A Forward Primer 20 nmol 1000 InfA-R SO3284 Influenza A Reverse Primer 20 nmol 1000 InfA-P SO3285 Influenza A Probe (Fam) 5 nmol 1000 InfB-F SO3311 Influenza B Forward Primer 20 nmol 1000 InfB-R SO3299 Influenza B Reverse Primer 20 nmol 1000 InfB-P SO3300 Influenza B Probe (Fam) 5 nmol 1000 RP-F SO3313 Human RNase P Forward Primer 20 nmol 1000 RP-R SO3314 Human Rnase P Reverse Primer 20 nmol 1000 RP-P SO3315 Human Rnase P Probe (Fam) 5 nmol 1000 Box #2: Pooled Influenza Positive Control and Human Specimen Extraction Control Reagent Label Part # Description Quantity / Tube Notes PIPC MR-088 Pooled Influenza Positive Control (PIPC) For use as a positive control with the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel procedure to ensure the detection of seasonal influenza virus A/H1, A/H3, A/H1pdm09 and influenza type B. The PIPC contains noninfectious positive control materials supplied as a liquid, 500 µl per vial, suspended in 0.01 M phosphate buffer saline (PBS) at ph 7.2-7.4. PIPC consists of four (4) different beta-propiolactone treated influenza viruses (influenza A/H1, A/H3, A/H1pdm09, and influenza B) and cultured human cells (A549). PIPC will yield a positive result with the following primer and probe sets: InfA, InfB, H1, H3, pdminfa, pdmh1, and RP. 1 tube x 500 µl / tube One thousand 5 µl reactions per tube Human Specimen Control (HSC) HS0096 Human Specimen Control (HSC): For use as an RNA extraction procedural control to demonstrate successful recovery of RNA as well as extraction reagent integrity. Purified RNA from the HSC material should yield a positive result with the RP primer and probe set and negative results with all influenza specific markers. The HSC consists of noninfectious (beta propiolactone treated) cultured human cell material supplied as a liquid suspended in 0.01 M PBS at ph 7.2-7.4. 17 tubes x 500 µl / tube Five 100 µl extractions per tube 7

Materials Required (But Not Provided) rrt-pcr Enzyme Mastermix Options Reagent Quantity Catalog No. Invitrogen SuperScript III Platinum One-Step Quantitative RT-PCR System (without Rox) Invitrogen SuperScript III Platinum One-Step Quantitative RT-PCR System (with Rox) Quanta BioSciences qscript One-Step qrt-pcr Kit, Low ROX Quanta BioSciences qscript One-Step qrt-pcr Kit, Low ROX 100 reactions 11732-020 500 reactions 11732-088 100 reactions 11745-100 500 reactions 11745-500 50 reactions 95059-050 200 reactions 95059-200 RNA Extraction Options Instrument/Manufacturer Extraction Kit Catalog No. Roche MagNA Pure LC 2.0 Roche MagNA Pure Compact Roche MagNA Pure Compact QIAGEN QIAGEN QIAcube Total Nucleic Acid Kit Nucleic Acid Isolation Kit I RNA Isolation Kit DSP Viral RNA Mini Kit DSP Viral RNA Mini Kit 192 extractions: 03 038 505 001 32 extractions: 03 730 964 001 32 extractions: 04 802 993 001 50 extractions: 61904 50 extractions: 61904 8

Instrument/Manufacturer Extraction Kit Catalog No. EasyMAG Magnetic Silica (280133) EasyMAG Disposables (280135) biomérieux NucliSENS easymag (Automated magnetic extraction reagents sold separately) EasyMAG Lysis Buffer (280134) EasyMAG Lysis Buffer, 2 ml (200292) EasyMAG Wash Buffers 1,2, and 3 (280130, 280131, 280132) Biohit Pipette Tips (280146) CDC Approved Ancillary Reagents with the CDC Human Influenza Virus Real- Time RT-PCR Diagnostic Panel Specific lots for the ancillary reagents that are not manufactured under Quality System Regulations (Invitrogen SuperScript TM and Quanta BioSciences qscript TM ) will be qualified for use with the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel by the CDC Influenza Division. Any lots not specifically qualified by the CDC Influenza Division for use with the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel are not valid for use with this device, and may affect device performance. A supplemental cumulative list of the qualified ancillary reagents lots for use with the Flu rrt-pcr Dx Flu Panel is available and can be requested by sending an email to FluSupport@cdc.gov Any issues related to assay performance or test failure that are suspected to involve ancillary reagents should be reported to the CDC Influenza Division by emailing FluSupport@cdc.gov Equipment and Consumables Required (But Not Provided) Plasticware and consumables RNase/DNase-free 1.5 ml polypropylene microcentrifuge tubes 100% ethanol (EtOH) Disposable gloves Molecular grade water (RNase/DNase Free) -70 C and -20 C freezer(s) 9

4 C refrigerator 96-well cold block ABI 7500 Fast Dx Real-Time PCR Instrument (Applied Biosystems, Foster City, CA) ABI 7500 Fast Sequence Detection Consumables (Applied Biosystems, Foster City, CA). ABI MicroAmp Fast 8-tube strip 0.1 ml, cat #4358293, or ABI MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 ml, part #4346906, #4346907, or part #4366932 (alternate to 8-strip tubes) ABI MicroAmp Optical 8-cap strip, cat #4323032 (required, do not use film) Micropipettors (range between 1-10 µl, 10-200 µl and 100-1000 µl) Benchtop microcentrifuge Warnings and Precautions For in-vitro diagnostic use (IVD). Follow standard precautions. All patient specimens and positive controls should be considered potentially infectious and handled accordingly. Do not eat, drink, smoke, apply cosmetics or handle contact lenses in areas where reagents and human specimens are handled. Handle all specimens as if infectious using safe laboratory procedures. Refer to Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition BMBL (http://www.cdc.gov/biosafety/) for standard biological safety guidelines for all procedures. Specimen processing should be performed in accordance with national biological safety regulations. If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state health departments for testing IMMEDIATELY. Virus culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens. Note: Novel influenza A viruses are new or re-emergent human strains of influenza A that cause cases or clusters of human disease, as opposed to those strains commonly circulating in humans that cause seasonal epidemics and to which human populations have residual or limited immunity (either by vaccination or previous infection). Performance characteristics have been determined with human upper respiratory specimens (including NPS, NS, TS, NA, NW, DPS/TS) and lower respiratory tract specimens (such as BAL, BW, TA, sputum and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture. Perform all manipulations of live virus samples within a Class II (or higher) biological safety cabinet (BSC). Use personal protective equipment such as (but not limited to) gloves and lab coats when handling kit reagents while performing this assay and handling materials including samples, reagents, pipettes, and other equipment and reagents. Amplification technologies such as PCR are sensitive to accidental introduction of product from previous amplification reactions. Incorrect results could occur if either the clinical specimen or the real-time reagents used in the amplification step become contaminated by accidental introduction of amplification product (amplicon). Workflow in the laboratory should proceed in a unidirectional manner. 10

Maintain separate areas for assay setup and handling of nucleic acids. Always check the expiration date prior to use. Do not use expired reagent. Do not substitute or mix reagent from different kit lots or from other manufacturers. Change aerosol barrier pipette tips between all manual liquid transfers. During preparation of samples, compliance with good laboratory techniques is essential to minimize the risk of cross-contamination between samples, and the inadvertent introduction of nucleases into samples during and after the extraction procedure. Proper aseptic technique should always be used when working with nucleic acids. Maintain separate, dedicated equipment (e.g., pipettes, microcentrifuges) and supplies (e.g., microcentrifuge tubes, pipette tips) for assay setup and handling of extracted nucleic acids. Wear a clean lab coat and powder-free disposable gloves (not previously worn) when setting up assays. Change gloves between samples and whenever contamination is suspected. Keep reagent and reaction tubes capped or covered as much as possible. Primers, probes (including aliquots), and enzyme master mix must be thawed and maintained on cold block at all times during preparation and use. Work surfaces, pipettes, and centrifuges should be cleaned and decontaminated with cleaning products such as 5% bleach, DNAzap or RNase AWAY to minimize risk of nucleic acid contamination. Residual bleach should be removed using 70% ethanol. Reagents, master mix, and RNA should be maintained on cold block or on ice during preparation and use to ensure stability. Dispose of unused kit reagents and human specimens according to local, state, and federal regulations. Reagent Storage, Handling, and Stability Store all primers and probes at 2-8 C until re-hydrated for use; Store all control materials (HSC, PIPC) at -20 C. Always check the expiration date prior to use. Do not use expired reagents. Protect fluorogenic probes from light. Primers, probes (including aliquots), and enzyme master mix must be thawed and kept on cold block at all times during preparation and use. Do not refreeze probes. Controls and aliquots of controls must be thawed and kept on ice at all times during preparation and use. 11

Specimen Collection, Handling, and Storage Inadequate or inappropriate specimen collection, storage, and transport are likely to yield false negative test results. Training in specimen collection is highly recommended due to the importance of specimen quality. CLSI MM13-P may be referenced as an appropriate resource. Collecting the Specimen Follow specimen collection devices manufacturer instructions for proper collection methods. Swab specimens should be collected using only swabs with a synthetic tip, such as nylon or Dacron, and an aluminum or plastic shaft. Calcium alginate swabs are unacceptable and cotton swabs with wooden shafts are not recommended. Transporting Specimens Ensure that, when transporting human respiratory specimens, all applicable regulations for the transport of etiologic agents are met. Human respiratory specimens, to be tested within 72 hours post-collection, should be transported refrigerated at 2 8 C. Alternatively, specimens may be frozen and transported for testing. Respiratory specimens should be collected and placed into viral transport media (VTM) as described by CDC and WHO guidelines. For more information refer to WHO Guidelines for the collection of clinical specimens during field investigation of outbreaks, http://www.who.int/csr/resources/publications/surveillance/whocdscsredc2004.pdf, and/or www.cdc.gov/h1n1flu/specimencollection.htm. Storing Specimens Specimens received cold should be stored refrigerated (2 8 C) for up to 72 hours before processing. Store any residual specimens at -70 C. Although optimal performance is met when testing fresh specimens within 72 hours of collection, performance has been demonstrated with frozen specimens. If testing of a fresh specimen is not possible within 72 hours storage at 2 8 C, the specimen may be frozen at -70 C and tested at a later time. Specimens received frozen should be stored at -70 C until processing. Store any residual specimens at -70 C. Storing Purified Nucleic Acid Store purified nucleic acids at -70 C. Specimen Referral to CDC Referring a specimen to the CDC Ship all specimens and related RNA overnight to CDC. Ship frozen specimens on dry ice and non-frozen specimens on cold packs. Ship extracted RNA on dry ice. Refer to the International Air Transport Association (IATA - www.iata.org) for requirements for shipment of human or potentially infectious biological specimens. Prior to shipping, notify CDC Influenza Division (see contact information below) that you are sending specimens. Fill out the Influenza Specimen Submission Form completely include specimen type and Ct values detected by your laboratory. 12

Send all samples to the following recipient: Virology, Surveillance and Diagnosis Branch, Influenza Division Centers for Disease Control and Prevention c/o STAT, MS G-16 Attention: Dr. Stephen Lindstrom 1600 Clifton Rd., Atlanta, GA 30333 Phone: (404) 639-1587 or (404) 639-3591 Fax: (404) 639-0080 The emergency contact number for CDC Emergency Operations Center (EOC) is 770-488-7100. Please see the following WHO reference for more details: http://www.who.int/csr/resources/publications/surveillance/cds_epr_aro_2006_1.pdf Primer and Probe Preparation: Reagent and Controls Preparation 1. Upon receipt, store lyophilized primers and probes at 2-8 C. 2. Rehydration a. Remove primers and probes from 2-8 C. b. Allow primers and probes to sit at ambient temperature for 15 minutes. c. Quickly centrifuge dried primers and probes to collected pellet in the bottom of the tube. d. Pipette 0.5 ml (500 µl) of 10 mm Tris, ph 7.4-8.2 or PCR-grade water into each dried PCR primer or probe. e. Allow primers and probes to fully rehydrate for at least 15 minutes at room temperature. f. After primers and probes are fully rehydrated, pulse vortex to ensure a homogenous solution. 3. Prepare a Combined Oligonucleotide Mix for Each Marker Set a. For each marker set (e.g. InfA), pipette the entire volume (0.5 ml) of the reconstituted forward and reverse primers and probe into a single new, nucleasefree, sterile tube (henceforth, the primer/probe mix). b. Pulse vortex the combined primer/probe mix to ensure a homogenous solution. 4. Aliquot a. Label five new, nuclease-free, sterile tubes for each combined primer/probe mix with the following information: Primer or probe name Kit lot # Expiration date b. Aliquot 300 µl of each combined primer/probe mix into respective labeled tubes and store at -20 C. 13

5. Storage a. After rehydration i. Aliquots of primers and probes are stored at -20 C or below until expiration date as long as QC requirements are met. ii. Thawed aliquots of primers and probes may be stored at 2-8 C for up to 3 months. Combined primers/probe mix aliquots should be stored in the dark. Human Specimen Control (HSC) Preparation: 1. Reagent a. Noninfectious cultured human cell material supplied as a liquid suspension in 0.01 M PBS. b. Volume: 0.5 ml per vial. 2. Storage a. Store at -20 C or below upon receipt. Do not dilute. 3. Procedure a. Human Specimen Control must be extracted and processed with each batch of samples to be tested. The final volume of eluted RNA should equal the volume of extracted control material. For example, 100 µl of control material should result in 100 µl of RNA extract. b. Do not dilute extracted RNA prior to testing. Pooled Influenza Positive Control (PIPC) Preparation: 1. Reagent a. Inactivated, noninfectious influenza virus preparation supplied as a liquid suspension in 0.01 M PBS. b. Control contains four different influenza viruses representing influenza A/H1, A/H3, A/H1pdm09, and influenza B viruses in cultured human cells. c. Volume: 0.5 ml yields approximately 5 ml of positive control RNA. 2. Storage a. Store at -20 C or below upon receipt. Do not dilute. 3. Procedure a. PIPC must be extracted prior to use. The final volume of eluted RNA should equal the volume of extracted control material. For example, 100 µl of control material should result in 100 µl of RNA extract (1:1). b. Dilute the RNA 1:10 with nuclease free water. c. Label one new nuclease-free, sterile, tube for each single-use aliquot with the following information: Control RNA name and 1:10 dilution Kit lot # Expiration date d. Dispense the 1:10 diluted RNA into single-use aliquots and store at -20 C or below for up to 6 months. Recommended aliquot volume (but not required): 20 µl aliquots for use with the Influenza A/B Typing Kit screening assays. e. Use one aliquot per run. Discard after use. Do not use residual RNA. 14

General Preparation Equipment Preparation Clean and decontaminate all work surfaces, pipettes, centrifuges, and other equipment prior to use. Decontamination agents should be used including 5% bleach, 70% ethanol, and DNAzap or RNase AWAY to minimize the risk of nucleic acid contamination. Reagent Preparation NOTE: All reagents should be kept on ice or cold rack during assay preparation. Primers and Probes Reagents Thaw frozen aliquots of primer/probe mix. Thawed aliquots may be stored at 2-8 C in the dark for up to 3 months. Do not re-freeze. Vortex each combined primer/probe mix for 15 seconds. Briefly centrifuge each primer/probe mix. Place combined primer/probe mix in cold rack during master mix preparation. Real-time RT-PCR Reagents (Important: Select Appropriate Enzyme System) Invitrogen SuperScript III Platinum One-Step Quantitative RT-PCR System Place Invitrogen 2X PCR Master Mix and Superscript III RT/Platinum Taq enzyme mix in a cold rack at 2-8 C. Completely thaw the 2X PCR Master Mix vial. Mix the 2X PCR Master Mix by inversion 10 times. Briefly centrifuge 2X PCR Master Mix and Superscript III RT/Platinum Taq enzyme mix then place in cold rack. OR Quanta BioSciences qscript One-Step qrt-pcr Kit, Low ROX Place Quanta qscript One-Step Master Mix (2X) and qscript One-Step Reverse Transcriptase in a cold rack at 2-8 C. Completely thaw the One-Step Master Mix (2X) vial. Mix the One-Step Master Mix (2X) by inversion 10 times. Briefly centrifuge One-Step Master Mix (2X) and qscript One-Step Reverse Transcriptase then place in cold rack. Nucleic Acid extraction Performance of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel is dependent upon the amount and quality of template RNA purified from human specimens. The following commercially available RNA extraction kits and procedures have been qualified and validated for recovery and purity of RNA for use with the panel: Qiagen QIAamp DSP Viral RNA Mini Kit Recommendation(s): Utilize 100 μl of sample and elute with 100 μl of buffer, or utilize 140 μl of sample and elute with 140 μl of buffer. 15

Qiagen QiaCube with the QIAamp DSP Viral RNA Mini Kit Kit: Qiagen QIAamp DSP Viral RNA Mini Kit Recommendations: Utilize 140 μl of sample and elute with 100 μl of buffer. Roche MagNA Pure LC Kit: Roche MagNA Pure Total Nucleic Acid Kit Protocol: Total NA External_lysis Recommendation(s): Utilize 100 μl of sample and 300 μl of lysis buffer (total sample volume for input into LC is 400 μl). Elution volume is 100 μl. These volumes should be available on the LC software. Roche MagNA Pure Compact Kit: Roche MagNA Pure Nucleic Acid Isolation Kit I Protocol: Total_NA_Plasma100_400 Recommendation(s): Utilize 100 μl of sample and 300 μl of lysis buffer (total sample volume for input into Compact is 400 μl). Elution volume is 100 μl. Roche MagNA Pure Compact Kit: Roche MagNA Pure RNA Isolation Kit Protocol: RNA_Tissue Protocol Recommendation(s): Utilize 100 μl of sample and 250 μl of lysis buffer (total sample volume for input into Compact is 350 μl). Elution volume is 100 μl. biomérieux NucliSENS easymag Instrument Protocol: General protocol (not for blood) using Off-board Lysis reagent settings Recommendation(s): Utilize 100 μl of sample and 1000 μl of lysis buffer (total sample volume for input into Compact is 1100 μl). Incubate for 10 minutes at room temperature. Elution volume is 100 μl. Manufacturer s recommended procedures are to be used for sample extraction. Disclaimer: Names of vendors or manufacturers are provided as examples of suitable product sources. Inclusion does not imply endorsement by the Centers for Disease Control and Prevention. Assay Setup IMPORTANT INFORMATION! The CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel consists of individual assays that can be utilized to meet laboratory testing algorithms, for example to perform high throughput screening using the Influenza A/B Typing Assay. However, CDC strongly recommends that laboratories perform subtype characterization of all the influenza A positives with the Influenza A Subtyping Assay. Current recommendations can be found on the FluSupport SharePoint site at https://partner.cdc.gov/sites/ncird/fs/default.aspx. 16

Master Mix Preparation / Plate Setup 1. In the assay preparation area, label a sterile, nuclease-free, 1.5 ml tube for each reaction master mix to be prepared for each specific master mix / marker to be prepared (i.e. InfA, InfB, etc.). 2. Determine the number of reactions (N) to be prepared per assay. 3. Calculate the amount of each reagent to be added to the tube for each master mix by multiplying the number of reactions (samples plus controls) per marker by the volume of reagent indicated in Figure 1. NOTE: It is necessary to make excess reaction master mix to allow for pipetting error. Example: If number of samples (n) including controls = 1 to 14, then N = n + 1 If number of samples (n) including controls > 15, then N = n + 2 17

Figure 1. Steps and Calculations for Master Mix Preparation (Important: Select Appropriate Enzyme System) Invitrogen SuperScript III Platinum One-Step Quantitative RT-PCR System Step # Reagent Vol. of Reagent Added per Reaction 1 Nuclease-free Water N x 5.5 µl 2 Combined Primer/Probe Mix N x 1.5 µl 3 SuperScript III RT/Platinum Taq Mix N x 0.5 µl 4 2X PCR Master Mix N x 12.5 µl Total Volume N x 20.0 µl OR Quanta BioSciences qscript One-Step qrt-pcr Kit, Low ROX Step # Reagent Vol. of Reagent Added per Reaction 1 Nuclease-free Water N x 5.5 µl 2 Combined Primer/Probe Mix N x 1.5 µl 3 Quanta qscript One-Step Reverse Transcriptase N x 0.5 µl 4 One-Step Master Mix (2X) N x 12.5 µl Total Volume N x 20.0 µl 4. In a sterile labeled 1.5 ml tube, prepare master mix for each marker set to be tested by first calculating the amount of each reagent to be added for each primer/probe set reaction master mix required for the test being performed. 5. In the assay preparation area, dispense reagents into each respective labeled 1.5 ml microcentrifuge tube. After addition of the water, mix reaction mixtures by pipetting up and down. Do not vortex. 6. Centrifuge for 5 seconds to collect contents at the bottom of the tube, and then place the tube in a cold rack. 7. Set up reaction strip tubes or plates in a 96-well cooler rack. 8. Dispense 20 µl of each master mix into the appropriate wells going across the row as shown below (Figure 2): 18

Figure 2. Influenza A/B Typing Assay: Example of Reaction Master Mix Plate Set-up 1 2 3 4 5 6 7 8 9 10 11 12 A InfA InfA InfA InfA InfA InfA InfA InfA InfA InfA InfA InfA B InfB InfB InfB InfB InfB InfB InfB InfB InfB InfB InfB InfB C RP RP RP RP RP RP RP RP RP RP RP RP D empty empty empty empty empty empty empty empty empty empty empty empty E empty InfA InfA InfA InfA InfA InfA InfA InfA InfA InfA empty F empty InfB InfB InfB InfB InfB InfB InfB InfB InfB InfB empty G empty RP RP RP RP RP RP RP RP RP RP empty H empty empty empty empty empty empty empty empty empty empty empty empty 9. Prior to moving to the nucleic acid handling area, prepare the NTC reactions for column #1 in the assay preparation area. 10. Pipette 5 µl of nuclease-free water into the NTC sample wells. Securely cap NTC wells before proceeding. 11. Cover the entire reaction plate and move the reaction plate to the specimen nucleic acid handling area. Template Addition 1. Gently vortex nucleic acid sample tubes for approximately 5 seconds. 2. After centrifugation, place extracted nucleic acid sample tubes in the cold rack. 3. Samples should be added to column 2-11 (column 1, 11 (bottom half), and 12 are for controls) to the specific assay that is being tested as illustrated in Figure 3. Carefully pipette 5.0 µl of the first sample into all the wells labeled for that sample (i.e. Sample S1 down column #2). Keep other sample wells covered during addition. Change tips after each addition. 4. Securely cap the column to which the sample has been added to prevent cross contamination and to ensure sample tracking. 5. Change gloves often and when necessary to avoid contamination. 6. Repeat steps #3 and #4 for the remaining samples. 7. Add 5 µl of Human Specimen Control (HSC) extracted sample to the HSC wells (Figure 3, column 11). Securely cap wells after addition. This is applicable to any plate set-up. 8. Cover the entire reaction plate and move the reaction plate to the positive template control handling area. 19

Assay Control Addition 1. Pipette 5 µl of PIPC RNA to the sample wells of column 12(Figure 3). Securely cap wells after addition of the control RNA. NOTE: If using 8-tube strips, label the TAB of each strip to indicate sample position. DO NOT LABEL THE TOPS OF THE REACTION TUBES! 2. Briefly centrifuge reaction tube strips for 10-15 seconds. After centrifugation return to cold rack. NOTE: If using 96-well plates, centrifuge plates for 30 seconds at 500 x g, 4 C. Figure 3. Influenza A/B Typing Kit: Example of Sample and Control Set-up 1 2 3 4 5 6 7 8 9 10 11 12 A NTC S1 S3 S5 S7 S9 S11 S13 S15 S17 S19 PIPC B NTC S1 S3 S5 S7 S9 S11 S13 S15 S17 S19 PIPC C NTC S1 S3 S5 S7 S9 S11 S13 S15 S17 S19 PIPC D empty empty empty empty empty empty empty empty empty empty empty empty E empty S2 S4 S6 S8 S10 S12 S14 S16 S18 HSC empty F empty S2 S4 S6 S8 S10 S12 S14 S16 S18 HSC empty G empty S2 S4 S6 S8 S10 S12 S14 S16 S18 HSC empty H empty empty empty empty empty empty empty empty empty empty empty empty Create a Run Template on the ABI 7500 Fast Dx Real-time PCR Instrument (Required if no template exists) If the template already exists on your instrument please proceed to the RUNNING A TEST section. 1. Launch the ABI 7500 Fast Dx Real-time PCR System by double clicking on the ABI 7500 Fast Dx System icon on the desktop. 2. A new window should appear, select Create New Document from the menu. 20

Figure 4. New Document Wizard Window Make sure to change Run Mode to STANDARD 7500 3. The New Document Wizard screen in Figure 4 will appear. Select: a. Assay: Standard Curve (Absolute Quantitation) b. Container: 96-Well Clear c. Template: Blank Document d. Run Mode: Standard 7500 e. Operator: Your Name f. Comments: SDS v1.4 g. Plate Name: Your Choice 4. After making selections click Next at the bottom of the window. 21

Figure 5. Creating New Detectors 5. After selecting next, the Select Detectors screen (Figure 5) will appear. 6. Click the New Detector button (see Figure 5). 7. The New Detector window will appear (Figure 6). A new detector will need to be defined for each influenza primer and probe set. Creating these detectors will enable you to analyze each primer and probe set individually at the end of the reaction. Figure 6. New Detector Window 22

8. Start by creating the InfA Detector. Include the following: a. Name:InfA b. Description: leave blankc. c. Reporter Dye: FAM d. Quencher Dye: (none) e. Color: to change the color of the detector indicator do the following: Click on the color square to reveal the color chart Select orange as the color by clicking on the orange square After selecting color click OK to return to the New Detector screen f. Click the OK button of the New Detector screen to return to the screen shown in Figure 5. 9. Repeat step 6-8 for each influenza target in the A/B Typing Kit. Name Reporter Dye Quencher Dye InfA FAM (none) InfB FAM (none) RP FAM (none) 10. After each Detector is added, the Detector Name, Description, Reporter and Quencher fields will become populated in the Select Detectors screen (Figure 7). 11. Before proceeding, the newly created detectors must be added to the document. To add the new detectors to the document, click ADD (see Figure 7). Detector names will appear on the right hand side of the Select Detectors window (Figure 7). Figure 7. Adding New Detectors to Document LB-YYY Rev 0 DRAFT DO NOT DISTRIBUTE CDC/OID/NCIRD/Influenza Division 23

12. Once all detectors have been added, select (none) for Passive Reference at the top right hand drop down menu (Figure 8). Figure 8. Select Passive Reference Passive reference should be set to (none) as described above. 13. Click Next at the bottom of the Select Detectors window to proceed to the Set Up Sample Plate window (Figure 8). 14. In the Set Up Sample Plate window (Figure 9), use your mouse to select row A from the lower portion of the window, in the spreadsheet (see Figure 9). 15. In the top portion of the window, select detector InfA. A check will appear next to the detector you have selected (Figure 9). You will also notice the row in the spreadsheet will be populated with a colored U icon to indicate which detector you ve selected. 16. Repeat step 14-15 for each detector that will be used in the assay. 24

Figure 9. Sample Plate Set-up 17. Select Finish after detectors have been assigned to their respective rows. (Figure 10). Figure 10. Finished Sample Plate Set-up 18. After clicking Finish, there will be a brief pause allowing the ABI 7500 Fast Dx to initialize. This initialization is followed by a clicking noise. Note: The machine must be turned on for initialization. 19. After initialization, the Plate tab of the Setup (Figure 11) will appear. 25

20. Each well of the plate should contain colored U icons that correspond with the detector labels that were previously chosen. To confirm detector assignments, select Tools from the file menu, then select Detector Manager. Figure 11. Plate Set-up Window 21. The Detector Manager window will appear (Figure 12). Figure 12. Detector Manager Window 26

22. Confirm all influenza detectors are included and that each influenza target has a Reporter set to FAM and the Quencher is set to (none). 23. If all detectors are present, select Done. The detector information has been created and assigned to wells on the plate. Defining the Instrument Settings (Important: Use Appropriate Settings for Invitrogen SuperScript III or Quanta qscript Enzyme) 1. After detectors have been created and assigned, proceed to instrument set up. 2. Select the Instrument tab to define thermal cycling conditions. 3. Modify the thermal cycling conditions as follows (Figure 13): Invitrogen SuperScript III Platinum One-Step Quantitative RT-PCR System OR a. In Stage 1, Set to 30 min at 50 C; 1 Rep. b. In Stage 2, Set to 2.0 min at 95 C; 1 Rep. c. In Stage 3, Step 1 set to 15 sec at 95 C. d. In Stage 3, Step 2 set to 30 sec at 55.0 C. e. In Stage 3, Reps should be set to 45. f. Under Settings (Figure 13), bottom left-hand box, change volume to 25 µl. g. Under Settings, Run Mode selection should be Standard 7500. h. Step 2 of Stage 3 should be highlighted in yellow to indicate data collection (see Figure 13). Quanta BioSciences qscript One-Step qrt-pcr Kit, Low ROX a. In Stage 1, Set to 30 min at 50 C; 1 Rep. b. In Stage 2, Set to 5.0 min at 95 C; 1 Rep. c. In Stage 3, Step 1 set to 15 sec at 95 C. d. In Stage 3, Step 2 set to 30 sec at 55.0 C. e. In Stage 3, Reps should be set to 45. f. Under Settings (Figure 13), bottom left-hand box, change volume to 25 µl. g. Under Settings, Run Mode selection should be Standard 7500. h. Step 2 of Stage 3 should be highlighted in yellow to indicate data collection (see Figure 13). 27

Figure 13. Instrument Window 4. After making changes to the Instrument tab, the template file is ready to be saved. To save the template, select File from the top menu, then select Save As. Since the two enzyme options have different instrument settings it is recommended that the template be saved with a name indicating the enzyme option. 5. Save the template as Influenza AB Typing Kit SuperScript or Influenza AB Typing Kit qscript as appropriate in the desktop folder labeled ABI Run Templates (you must create this folder). Save as type should be SDS Templates (*.sdt) (Figure 14). 28

Figure 14. Saving Template Running a Test 1. Turn on the ABI 7500 Fast Dx Real-Time PCR Instrument. 2. Launch the ABI 7500 Fast Dx Real-time PCR System by double clicking on the 7500 Fast Dx System icon on the desktop. 3. A new window should appear, select Open Existing Document from the menu. 4. Navigate to select your ABI Run Template folder from the desktop. 5. Double click on the appropriate template file (Influenza AB Typing Kit SuperScript or Influenza AB Typing Kit qscript). 6. There will be a brief pause allowing the ABI 7500 Fast Dx Real-Time PCR Instrument to initialize. This initialization is followed by a clicking noise. Note: The machine must be turned on for initialization. 29

Figure 15. Plate Set-up Window 7. After the instrument initializes, a plate map will appear (Figure 15). The detectors and controls should already be labeled as they were assigned in the original template. 8. Click the Well Inspector icon from the top menu. 9. Highlight specimen wells of interest on the plate map. 10. Type sample identifiers to Sample Name box in the Well Inspector window (Figure 16). 30

Figure 16. Labeling Wells 11. Repeat steps 9-10 until all sample identifiers are added to the plate setup. 12. Once all specimen and control identifiers are added click the Close button on the Well Inspector window to return to the Plate set up tab. 13. Click the Instrument tab at the upper left corner. 14. The reaction conditions, volumes, and type of 7500 reaction should already be loaded. (Figure 17). 31

Figure 17. Instrument Settings 15. Ensure settings are correct (refer to the Defining Instrument Settings). 16. Before proceeding, the run file must be saved; from the main menu, select File, then Save As. Save in appropriate run folder designation. 17. Load the plate into the plate holder in the instrument. Ensure that the plate is properly aligned in the holder. 18. Once the run file is saved, click the Start button. Note: The run should take approximately 1hr and 45 minutes to complete 32

c. Data Analysis 1. After the run has completed, select the Results tab at the upper left corner of the software. 2. Select the Amplification Plot tab to view the raw data (Figure 18). Figure 18. Amplification Plot Window b c d e. a 3. Start by highlighting all the samples from the run; to do this, click on the upper left hand box (a) of the sample wells (Figure 18). All the growth curves should appear on the graph. 4. On the right hand side of the window (b), the Data drop down selection should be set to Delta Rn vs. Cycle. 5. Select InfA from (c), the Detector drop down menu, using the downward arrow. a. Please note that each detector is analyzed individually to reflect different performance profiles of each primer and probe set. 6. In the Line Color drop down (d), Detector Color should be selected. 7. Under Analysis Settings select Manual Ct (e). a. Do not change the Manual Baseline default numbers. 8. Using the mouse, click and drag the red threshold line until it lies within the exponential phase of the fluorescence curves and above any background signal (Figure 19). 33

Figure 19. Amplification Plot Exponential PCR Phase Background noise Threshold adjusted to fall within the PCR exponential phase. 9. Click the Analyze button in the lower right corner of the window. The red threshold line will turn to green, indicating the data has been analyzed. 10. Repeat steps 5-9 to analyze results generated for each set of markers (i.e. InfA, InfB, etc). 11. Save analysis file by selecting File then Save As from the main menu. 12. After completing analysis for each of the markers, select the Report tab above the graph to display the Ct values. To filter report by sample name in ascending or descending order, simply click on Sample Name in the table. Figure 20. Report 34

Interpretation of Results and Reporting Extraction and Positive Control Results and Interpretation No Template Control (NTC) The NTC consists of using nuclease-free water in the rrt-pcr reactions instead of RNA. The NTC reactions for all primer and probe sets should not exhibit fluorescence growth curves that cross the threshold line. If any of the NTC reactions exhibit a growth curve that crosses the cycle threshold, sample contamination may have occurred. Invalidate the run and repeat the assay with strict adherence to the guidelines. Pooled Influenza Positive Control (PIPC) The PIPC consists of four different influenza viruses representing influenza A/H1N1, A/H3N2, A/H1pdm09, and influenza B viruses suspended in cultured human cells (A549). Purified RNA from the PIPC will yield a positive result with the following primer and probe sets: InfA, InfB, H1, H3, pdminfa, pdmh1, and RP. Human Specimen Control (HSC) (Extraction Control) The HSC control consists of noninfectious cultured human cell (A549) material. The HSC is used as an RNA extraction procedural control to demonstrate successful recovery of RNA as well as extraction reagent integrity. Purified RNA from the HSC should yield a positive result with the RP primer and probe set and negative results with all influenza specific markers. Expected Performance of Controls Included in the Influenza A/B Typing Assay Control Type Positive Negative Extraction Internal Control Name PIPC NTC HSC Used to Monitor Substantial reagent failure including primer and probe integrity Reagent and/or environmental contamination Failure in lysis and extraction procedure InfA InfB RP + + + - - - - - + Expected Ct Values < 38.00 Ct None detected < 38.00 Ct If the controls do not exhibit the expected performance as described, the assay may have been set up and/or executed improperly, or reagent or equipment malfunction could have occurred. Invalidate the run and re-test. RNase P (Extraction Control) All clinical samples should exhibit fluorescence growth curves in the RNase P reaction that cross the threshold line within 38.00 cycles (< 38.00 Ct), thus indicating the presence of the human RNase P gene. Failure to detect RNase P in 35

any clinical specimens may indicate: Improper extraction of nucleic acid from clinical materials resulting in loss of RNA and/or RNA degradation. Absence of sufficient human cellular material due to poor collection or loss of specimen integrity. Improper assay set up and execution. Reagent or equipment malfunction. If the RP assay does not produce a positive result for human clinical specimens, interpret as follows: If the InfB or InfA are positive even in the absence of a positive RP, the influenza result should be considered valid. It is possible, that some samples may fail to exhibit RNase P growth curves due to low cell numbers in the original clinical sample. A negative RP signal does not preclude the presence of influenza virus RNA in a clinical specimen. If all influenza markers AND RNase P are negative for the specimen, the result should be considered inconclusive for the specimen. If residual specimen is available, repeat the extraction procedure and repeat the test. If all markers remain negative after re-test, report the results as inconclusive and a new specimen should be collected if possible. The RP assay may be negative when testing virus culture samples. Influenza Markers (InfA, InfB) When all controls exhibit the expected performance, a specimen is considered negative if all influenza marker (InfA, InfB) cycle threshold growth curves DO NOT cross the threshold line within 38.00 cycles (< 38.00 Ct) AND the RNase P growth curve DOES cross the threshold line within 38.00 cycles (< 38.00 Ct). When all controls exhibit the expected performance, a specimen is considered positive for influenza if the influenza marker (InfA, InfB) cycle threshold growth curve crosses the threshold line within 38.00 cycles (< 38.00 Ct). The RNase P may or may not be positive as described above, but the influenza result is still valid. When testing tissue culture derived samples, the Rnase P result is likely to yield negative / not detected result due to the absence of the human RNase P target. When all controls exhibit the expected performance and the growth curves for the influenza markers (InfA, InfB) AND the RNase P marker DO NOT cross the cycle threshold growth curve within 38.00 cycles (< 38.00 Ct), the result is inconclusive. The extracted RNA from the specimen should be re-tested. If residual RNA is not available, re-extract RNA from residual specimen and re-test. If the re-tested sample is negative for all markers and all controls exhibit the expected performance, the result is Inconclusive. 36

Influenza A/B Typing Assay Results Interpretation Guide The table below lists the expected results for the Influenza A/B Typing Assay. If results are obtained that do not follow these guidelines, re-extract and re-test the sample. If repeat testing yields similar results, contact CDC Technical Support for consultation and possible specimen referral as some results may indicate novel or emerging influenza viruses. See pages 12 and 55 for referral and contact information. Influenza A/B Typing Assay InfA InfB RP Result Interpretation a Report for CDC Surveillance Notes and Special Guidance + - ± Influenza A Detected; Subtyping not performed Influenza A - + ± Influenza B Detected Influenza B - - + Influenza NOT Detected Not Detected + + ± - - - Report: Influenza A Detected and Influenza B Detected; Refer to CDC for further characterization. Inconclusive Failure of controls Inconclusive Inconclusive If original result, re-extract and re-test. If repeat, report as possible co-infection. Specimen should be referred to CDC for further characterization; possible coinfection or LAIV detection If original result, re-extract and re-test. If repeat, report as Inconclusive. Obtain new specimen if possible. a Laboratories should report their diagnostic result as appropriate and in compliance with their specific reporting system. While this device can be used as a test for influenza A and B only, CDC strongly recommends that laboratories subtype all the influenza A positives with the Influenza A Subtyping Assay. The CDC recommends maintaining the enhanced surveillance efforts by state and local health departments, hospitals, and clinicians to identify patients that may be transmitting a possible novel or newly emerging influenza virus. 37